1. Field of the Invention
The invention relates to devices for the preparation of a plurality of sample sites on mass spectrometric sample supports, where samples are prepared with liquids in such a way that they are suitable for ionization in a mass spectrometer, for example ionization by matrix-assisted laser desorption (MALDI).
2. Description of the Related Art
The preparation of samples for ionization by matrix-assisted laser desorption (MALDI) requires aggressive organic solvents to dissolve the matrix material, and to apply the solutions to the sample support manually or with the aid of pipetting robots. The solvent must subsequently be vaporized to allow the growth of crystals of the matrix substance, into which likewise dissolved analyte substances are embedded. Since ionization by matrix-assisted laser desorption (MALDI) is widely known, no detailed description will be given here.
Nowadays, the mass-spectrometric identification of microbial samples involves daily preparation of many thousands of samples in microbiological laboratories for ionization by matrix-assisted laser desorption. Since only the substances from inside the microbes are useful for the mass-spectrometric identification, the cells of the microbes must first be cell disrupted. Often this also takes place on the sample support. The first step here is to apply small, hardly visible quantities of around 105 to 107 microbes from agar plate colonies onto the sample sites of the sample support. The cells of the microbes are usually cell disrupted by strong acids, which must subsequently be dried up by vaporization. Seventy percent formic acid (boiling point 101° C.; vapor pressure 43 hPa at 20° C.) or trifluoroacetic acid of similar concentration (TFA; boiling point 72° C.; vapor pressure 110 hPa at 20° C.) is usually used for this purpose. Quantities of around one microliter are applied onto each sample; once the acid is almost dried up, the matrix solution is applied, also in quantities of around one microliter. The matrix solution usually contains a solid organic acid (usually α-cyano-4-hydroxycinnamic acid, HCCA, but also 2,5-dihydroxybenzoic acid, DHB, for example) in a solvent mixture of acetonitrile and alcohols, for safety reasons usually ethanol, although methanol would be the better alternative. The matrix solution penetrates into the microbes through the weakened cell walls, causing them to burst and dissolving the soluble contents. The microbes are then identified with the aid of a mass spectrum of the contents.
The sample supports are usually the size of microtitration plates (or a fraction thereof) and nowadays usually have 96 or 384 visible sample sites for the application and preparation of the samples. Sample supports with 1536 sample sites are also in use. The sample sites with diameters of 0.8 to 2.0 millimeters can be identified with the aid of milled-in rings, where the milled edges prevent the applied acids and solvents from flowing away; the sample sites can also take the form of hydrophilic areas in a hydrophobic environment.
At present, the preparation is still mostly carried out manually with dispensing pipettes, without a hood (fume cupboard) because hoods are rare in microbiological laboratories. This can be hazardous to health if the ventilation is insufficient. Even when a hood is available, it is often not used because pipetting in an open hood is very awkward. So there has long been a need for an automatic preparation device which facilitates the application of the acids and matrix solutions, does not release hazardous vapors, and preferably does not require a hood.
The liquid quantities of around one microliter do not drip from the pipette tip of their own accord, but must be applied according to the Prior Art by dabbing them onto the sample. A new pipette tip must be used for each sample in order to prevent samples being transferred. Non-contact application of the liquids onto the samples would therefore be particularly advantageous because it would eliminate the need to replace the pipette tips each time.
For non-contact application it is advantageous to position the dispenser exactly vertically above the sample sites. Automatic preparation thus requires the provision of movement devices which can bring the dispenser and the sample sites precisely in line vertically. Since the evaporating acids and solvents attack surfaces, especially metal surfaces in sliding contact, the usual cross slides driven by stepper motors and screw threads (as with an X-Y translation stage) cannot be used.
This disclosure references ionization by matrix-assisted laser desorption (MALDI), in which ions are produced during the desorption process initiated by laser beam pulses. It goes without saying, however, that sample preparations shall also be possible where the analyte substances in the prepared samples can be transferred into the gaseous phase regardless of their ionization. The type of ionization can be selected as required to suit the application. Simple laser desorption in combination with chemical ionization (LDCI) can be carried out, for example, or direct electrospray ionization from the surface (DESI), but other types of ionization can also be used. Accordingly, the term “ionization with matrix-assisted laser desorption” must not be understood as a restriction.
Furthermore, this disclosure occasionally mentions the application of a drop onto a sample. It is understood that the dispensed drop can also be applied to an uncoated sample site of the sample support, for example as a preparatory step for the subsequent coating with a microbial sample. Accordingly, embodiments of this type, also, must not be deemed to be in any way restrictive.
In view of the foregoing, there is a need for a device which can automatically prepare the sample sites on mass-spectrometric sample supports with liquids for a later ionization of the samples, for example for an ionization by matrix-assisted laser desorption, with strongly reduced effect of released vapors on the movement devices. It is preferable if the device is designed in such a way that the user cannot be endangered by the vapors given off either.